温肺化纤汤对过氧化氢诱导的肺间充质干细胞氧化应激损伤模型有氧糖酵解功能的影响

Effect of Wenfei Huaxian Decoction(温肺化纤汤)on Aerobic Glycolysis of Hydrogen Peroxide-induced Oxi⁃dative Stress Injury of Lung Mesenchymal Stem Cells in Mice

目的探讨温肺化纤汤治疗特发性肺纤维化(IPF)其可能机制。方法梯度密度离心法分离提取C57BL/6小鼠肺间充质干细胞(LMSCs)。体外培养LMSCs,采用不同浓度(100、200、300、400、500、600、700、800μmol/L)的过氧化氢(H_(2)O_(2))诱导细胞损伤,MTT法检测LMSCs细胞存活率,选择细胞存活率约为50%的H_(2)O_(2)损伤浓度进行后续试验。细胞分为正常对照组、温肺化纤汤组,温肺化纤汤组设浓度为1600、800、400、200、100、50、25μg/ml,MTT法检测温肺化纤汤的毒性。细胞分为正常对照组、模型组、温肺化纤汤组,温肺化纤汤设浓度为1600、800、400、200、100、50、25μg/ml,根据剂量最小化和疗效最大化的原则,结合细胞存活率确定药物的最佳浓度用于后续试验。实验分为正常对照组、模型组、阳性对照组及温肺化纤汤低、中、高剂量组。除正常对照组外,模型组和温肺化纤汤各剂量组分别在温肺化纤汤预处理24 h后均给予H_(2)O_(2)造模6 h。造模结束后检测细胞上清中葡萄糖摄取量及乳酸水平,RT-PCR检测有氧糖酵解相关酶己糖激酶2(HK2)、丙酮酸激酶2(PKM2)mRNA水平。结果600μmol/L H_(2)O_(2)作用于LMSCs 6 h,细胞的存活率为(52.17±0.82)%,并作为后续氧化应激损伤模型的浓度。与正常对照组比较,不同浓度的温肺化纤汤(25~1600μg/ml)对LMSCs均无毒性作用(P>0.05)。与模型组比较,不同浓度(25~1600μg/ml)温肺化纤汤预处理的LMSCs存活率均显著增加(P<0.01);按4倍设定等比浓度梯度,采取终浓度为25、100、400μg/ml温肺化纤汤作为低、中、高剂量组的干预浓度。与正常对照组相比,模型组LMSCs氧化应激损伤后导致葡萄糖摄取量和乳酸含量降低(P<0.01),有氧糖酵解相关酶HK2和PKM2 mRNA水平均下降(P<0.05)。与模型组对比较比,温肺化纤汤各剂量组均可增强葡萄糖摄取量和乳酸的水平(P<0.05),且温肺化纤汤中、高剂量(100、400μg/ml)则增强了HK2、PKM2 mRNA表达(P<0.01)。与阳性对照组比较,温肺化纤汤高剂量组葡萄糖摄取量、乳酸含量均升高(P<0.05)。与温肺化纤汤低剂量组比较,温肺化纤汤中、高剂量组的HK2、PKM2 mRNA水平均明显增高(P<0.05)。结论温肺化纤汤可能通过增强LMSCs的有氧糖酵解改善H_(2)O_(2)诱导的细胞氧化应激损伤,从而治疗IPF。

Objective To investigate the possible mechanism of Wenfei Huaxian Decoction(温肺化纤汤,WHD)in the treatment of idiopathic pulmonary fibrosis(IPF).Methods C57BL/6 mice lung mesenchymal stem cells(LMSCs)were isolated by gradient density centrifugation.The LMSCs were cultured in vitro,and different concentra⁃tions(100,200,300,400,500,600,700 and 800μmol/L)of hydrogen peroxide(H_(2)0_(2))were used to induce cell injury.The survival rate of LMSCs was detected by MTT method,and the H_(2)0_(2) concentration corresponding to the sur⁃vival rate of 50%was used in subsequent experiments.The cells were divided into normal control group and WHD groups with different concentrations(1600,800,400,200,100,50,and 25μg/ml),and the MTT method was used to detect the toxicity of WHD.The cells were divided into normal control group,model group,and WHD groups with different concentrations(1600,800,400,200,100,50,and 25μg/ml),and the optimal concentration of WHD was determined for subsequent experiments following the principle of minimum dose with maximum effect and referring to the survival rate of the cells.The sample were divided into normal control group,model group,positive control group,and WHD low,medium and high dose groups.Except for the normal control group,the model group and all WHD dose groups were given H_(2)0_(2) for 6 hours 24 hours after pretreatment to develop the model.After model⁃ing,the glucose intake and the lactic acid level in cell supernatant were detected.The mRNA levels of the aerobic gly⁃colysis related hexokinase 2(HK2)and pyruvate kinase 2(PKM2)were detected by RT-PCR.Results When 600μmol/L H_(2)0_(2) were given to LMSCs for 6 h,the cell survival rate was(52.87±0.82)%.Therefore,600μmol/L was used as the concentration of H_(2)0_(2) in subsequent oxidative stress injury model.Compared to the normal control group,the WHD groups with different concentrations(25μg/ml to 1600μg/ml)all had no toxic effect on LMSCs(P>0.05).Compared to that in the model group,the cell survival rate of LMSCs in the WHD groups significantly in⁃creased after pretreatment with different concentrations(25μg/ml to 1600μg/ml)of WHD(P<0.01).The equal concentration gradient with ratio of 4∶1 were set,and the concentration of 25,100 and 400μg/ml were determined as the low,medium and high dose of WHD.Compared to those in the normal control group,the glucose uptake and lactic acid content in the model group decreased(P<0.01),and the mRNA levels of aerobic glycolysis related en⁃zymes HK2 and PKM2(P<0.05)after oxidative stress injury of LMSCs.Compared to the model group,all dose groups of WHD increased the glucose intake and lactic acid level(P<0.05),while the WHD medium and high dose groups(100 and 400μg/ml)increased the mRNA expression of HK2 and PKM2(P<0.01).Compared to the posi⁃tive control group,the WHD high dose group had higher glucose intake and lactic acid content(P<0.05).Com⁃pared to the low dose group of WHD,the medium and high dose groups significantly increased the mRNA levels of HK2 and PKM2(P<0.05).Conclusion WHD can improve the oxidative stress injury induced by H_(2)0_(2) in the treatment of IPF,and its mechanism may be related to enhancing the aerobic glycolysis of LMSCs.